Tannins have demonstrated antibacterial and antibiofilm activity, but there are still unknown aspects on how the chemical properties of tannins affect their biological properties. We are interested in understanding how to modulate the antibiofilm activity of tannins and in delineating the relationship between chemical determinants and antibiofilm activity.Salmonella Typhimurium, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus was determined in the Calgary biofilm device.The effect of five different naturally acquired tannins and their chemical derivatives on biofilm formation and planktonic growth of Most of the unmodified tannins exhibited specific antibiofilm activity against the assayed bacteria. The chemical modifications were found to alter the antibiofilm activity level and spectrum of the tannins. A positive charge introduced by derivatization with higher amounts of ammonium groups shifted the anti-biofilm spectrum toward Gram-negative bacteria, and derivatization with lower amounts of ammonium groups and acidifying derivatization shifted the spectrum toward Gram-positive bacteria. Furthermore, the quantity of phenolic OH-groups per molecule was found to have a weak impact on the anti-biofilm activity of the tannins.We were able to modulate the antibiofilm activity of several tannins by specific chemical modifications, providing a first approach for fine tuning of their activity and antibacterial spectrum. Plant-derived tannins have been used from ancient times in leather industry because of their ability of making leather last over time, and the name “tannin” comes from the use of these chemicals for “tanning” leather . AccordiFrom a molecular point of view, tannins can be divided in two main groups: condensed and hydrolysable tannins. Hydrolysable tannins are esters of gallic acid with a core sugar, often glucose or quinic acid. Tannic acid is the most prominent representative of the family of hydrolysable tannins comprising a glucose center . Condensvia covalent or non-covalent linking  in  catappa  and T. chebulata , and indnic acid . This tynic acid .Because of this reduced potential of resistance development, in the current study we evaluated selected chemical variants of tannins for their ability to inhibit biofilm formation without inhibiting planktonic growth. A diverse range of five commercially obtained tannins, three condensed tannins and two hydrolysable tannins, were included. The tannins were selected because they have been structurally thoroughly characterized in prior works and they are representative of the known molecular diversity of tannins. Also, we investigated how different chemical modifications change the activity level and spectrum of the tannins. Understanding the effect of structural features on activity allows to enhance or finetune activity. The functional groups chosen for derivatization were selected for known capacity (i) to interfere with membranes, i.e., ammonium groups; and (ii) to alter modes of interactions with surfaces, i.e., carboxylic acids and poly(ethylene glycol). The objective was to study the consequences of such chemical derivatizations at the molecular level and at the level of intermolecular interaction possibilities and obtain hints regarding matches and mismatches between tannin-type and tannin functionalization with respect to tuning anti-biofilm activities. The scarcity of structure–activity relationship (SAR) research in the field of tannins and bioactive phenolic compounds from plant sources as antimicrobial compounds highlights the value of this work .Vv-20), Omnivin WG (procyanidins (62%)/profisetidins (34%), Vv) and Mimosa ATO ME (prorobinetidins (33%)/profisetidins (67%), Am), and the two hydrolysable tannins were Tanal 01  and Tanal 04 . The chemical structures of these tannins were elucidated in detail as reported elsewhere  catechin, lsewhere . Figure via their phenolic functionalities, as reported in detail elsewhere , (ii) hydroxypropyl-1-carboxylic acid (C3COOH-eq), and (iii) oligomeric ethylene glycol polyether (PEG500-eq), whereby “eq.” in the compounds listed in 3NMe3Cl-eq added positive charges to the tannin molecule, (ii) C3COOH-eq, as a weak acid, potentially added negative charges to the molecule, and (iii) PEG500-eq polymerized the tannin molecules. During the various functionalizations, control tannins were re-isolated from blank reactions. Tannins labeled as “Blank-W” are tannins isolated from blank reactions performed in water and tannins labeled as “Blank-D” are tannins isolated from blank reaction preformed in dimethylformamide.The five selected tannins were chemically modified by derivatizing them lsewhere , with diBecause of the low solubility of the tannins in aqueous media, the dry compounds were first suspended in dimethyl sulfoxide (DMSO) at a stock concentration of 60 g/L and from there diluted to the desired concentration in the following experiments. A 1% v/v concentration of DMSO was never exceeded in order to prevent potential effects of DMSO on bacterial growth or biofilm formation.All the tests were done in aerobic conditions in growth media with a pH of ~7.4 and a salinity range of 0.025–0.5% w/v.The antibiofilm activities of the tannins were determined using 96-well plates with a cover lid with pegs for growing the bacterial biofilms in presence of several concentration of the assayed tannins. These assays, divided in a 1st exploratory screening (Section “Antibiofilm screening”) and a 2nd validation experiment , are explained in detail in the following section.Salmonella enterica var. Typhimurium ATCC14028, Pseudomonas aeruginosa PA14, Escherichia coli TG1 and Staphylococcus aureus SH1000 were grown in the Calgary biofilm device via a protocol that was previously described . The diluted ON cultures were then added to the wells to obtain a starting inoculum of 106 cfu/ml in a final volume of 200 μl of growth medium per well. Also, both for the exploratory screening test and validation of anti-biofilm activity, one row of the plate was filled with inoculum with growth media without tannin and another row was filled with media without bacteria. To account for potential effects of the compounds themselves, the same tannin-concentration was added to separate control 96-well Calgary biofilm plates without the inoculation of the bacteria. Afterwards, all plates were incubated in a wet chamber for the appropriate time and temperature: 48 h at 37°C for S. aureus, 24 h at 25°C for the Gram-negatives.Biofilms of escribed . The bac600 measurements of the base plate of the device, respectively. Specifically, after incubation the covers of the plates (which contain the pegs) were removed and washed once with PBS. The pegs were then stained with 200 μl per well of 0.1% v/v of crystal violet  for 30 min. After staining, the excess of CV was washed once with 200 μl per well of distilled water and let dry for 30 min. Finally, the CV was recovered in a new 96-well plate with 200 μl per well of 30% v/v glacial acetic and the optical density at 570 nm (OD570) measured in a plate reader. The optical density at 600 nm (OD600) of the bacteria in the base plates was measured to determine the growth of the planktonic cells.Biofilm formation and planktonic growth were determined by crystal violet staining of the pegs and OD570 (for the biofilm formation) and OD600 (for the planktonic growth) were corrected by the OD of the tannins incubated in absence of bacterial inoculum and then normalized using the average OD of the bacterial inoculum incubated in absence of tannin, thus being converted to percentage of biofilm formation (OD570) and percentage of bacterial growth (OD600).In a preliminary experiment, the tannins were tested in two-fold dilution series ranging from 600 to 9.38 mg/L in 3 independent replicates. The obtained raw OD50 and IC50  were calculated by applying a log [tannin] vs. percentage of biofilm formation or percentage of planktonic growth non-linear regression (four-parameters) using the statistical package GraphPad 8.0.The BICVv-20, Vv, Am, Ta-01, Ta-04; (ii) concentration of tannin (mg/L): 9.38, 79.69, 150; (iii) chemical modifier: C3COOH, PEG, C3NMe3Cl, Blank-D (blank reaction with dimethylformamide), Blank-W (blank reaction with water), unmodified; and (iv) concentration of applied chemical modifier: low and high . In order to minimize effects of plate-to-plate variation, we applied an optimal randomized experimental design with the statistical package JMP 15.0 to reduce experimental noise and confounding factors. Different to the preliminary experiment, only three concentrations were assayed, which were chosen to best capture the antibiofilm activity of the tannins: 9.38, 79.69, and 150 mg/L. The remaining part of the protocol of this experiment is identical to that of the preliminary experiment described above.Based on the information from the preliminary experiment, a definitive antibiofilm experiment in the Calgary biofilm device was set up with eight independent repeats. We first defined the factors under study, i.e., the parameters that potentially have an influence on the formation of biofilm. The considered parameters are: (i) original unmodified tannin: To determine the effect of the chemical derivatizations on the antibiofilm and antibacterial effect of the unmodified tannins on each of the assayed bacteria, an ANOVA test with Tukey post-hoc test comparing the unmodified tannins with their respective derivatized tannins was done based on the obtained biofilm formation and planktonic growth levels.via31PNMR as described elsewhere (To determine the effect of phenolic hydroxyl (OH) content on the antimicrobial and antibiofilm effect of the different tannins, a simple linear regression between the phenolic OH content of the tannins and the level of biofilm formation (or planktonic growth) was performed. To better calculate this correlation, we used the different tannin assayed concentrations and the obtained mmol of phenolic OH per gram of material to calculate the mmol of phenolic OH present in the system for each tannin at each assayed concentration, and we correlated this value with the respective percentage of biofilm inhibition and planktonic growth inhibition. The phenolic OH content of the tannins was determined lsewhere .Vv-20, (ii) the low oligomeric Vv and (iii) the higher oligomeric Am. The hydrolysable tannins consisted of two large tannins: (i) the “typical” tannic acid Ta-01 and (ii) the galloquinic acid derivative Ta-04. All tannins were previously functionalized with a positive charge introducing ammonium salt hydroxy-N,N,N-trimethylpropanyl-3-aminium chloride (C3NMe3Cl-0.1 and C3NMe3Cl-0.5), an acidifying hydroxypropyl-1-carboxylic acid (C3COOH-0.1 and C3COOH-0.1-0.5) motive and a polymerizing oligomeric ethylene glycol polyether  of the tannins were evaluated in order to obtain a first glance on activity spectrum and active concentration range. Activities against all four bacterial species were observed, with BIC50 values ranging from 4.69 to 545.8 mg/L and IC50 values ranging from 37.5 to 459.2 mg/L  catechins and fisetinidols  and cons9.2 mg/L . As suchlate see , S2.To allow a multivariate analysis considering tannin scaffold, derivatization and concentration as well as bacterial target species, the previous antibiofilm and antimicrobial experiments were repeated in one experiment with eight repeats per condition, but only focusing on the three tannin concentrations that could capture best the antibiofilm effect of the tannins: 9.38, 79.69, and 150 mg/L. In order to minimize previously observed effects of plate-to-plate variation, these experiments were designed in a randomized way, i.e., all the tannins were distributed through all the plates in a random fashion. This allowed to decrease the random error and the possibility of confounding factors, a necessary step for doing a complex statistical analysis that allows to link the different chemical characteristics. In what follows we will first focus on the unmodified tannins, after which we will elaborate on the effect of chemical derivatization.Staphylococcus aureus being the bacterium that reached the highest planktonic population density and formed most biofilm. On the contrary, the assayed Gram-negative bacteria showed less biofilm and less planktonic growth in comparison with S. aureus.To determine the percentual inhibition of biofilm formation and planktonic growth of the assayed tannins against the selected bacteria, the bacterial growth and biofilm development in absence of tannin was taken as reference. As can be seen in In Vv-20 exhibited significant antibiofilm activity against all the assayed bacteria at all the assayed concentrations in a clear dose-dependent way, a dose of 79.69 mg/L being sufficient for inhibiting biofilm formation more than 50% against the four assayed bacteria. Also, Vv-20 was the most effective tannin against S. Typhimurium, with more than 75% of biofilm inhibition at 79.69 and 150 mg/L, and against E. coli, with more than 80% biofilm inhibition at 79.69 mg/L. Regarding the low oligomeric Vv, the antibiofilm activity was preferential against P. aeruginosa and S. aureus, for which it exhibited potent dose-dependent antibiofilm activity. This compound was able to inhibit more than 50% of biofilm formation by S. aureus both at 79.69 and 150 mg/L, and is the unmodified tannin with highest effect against P. aeruginosa, displaying antibiofilm activity of more than 90% at 79.69 mg/L and more than 95% at 150 mg/L. On the contrary, Vv was less effective against S. Typhimurium and E. coli and was able to inhibit <30% of biofilm formation of both bacterial species, regardless of the assayed concentration. Contrary to the previous two unmodified condensed tannins, the high oligomeric Am had preferential antibiofilm activity against S. Typhimurium and S. aureus, but also had significant antibiofilm activity against P. aeruginosa. Moreover, Am is the unmodified tannin with the highest antibiofilm activity against S. aureus, with more than 85% of antibiofilm activity at 79.69 and 150 mg/L, also inhibiting more than 50% of biofilm formation of S. Typhimurium both at 79.69 and 150 mg/L, and inhibiting P. aeruginosa biofilm formation in more than 30% at 79.69 mg/L and more than 50% at 150 mg/L. On the contrary, Am had unnoticeable inhibitory activity against E. coli biofilms at any of the assayed concentrations.However, there were clear differences in the degree of antibiofilm activity of each tannin. Starting from the condensed tannins, the monomeric Ta-01 exhibited preferential activity against P. aeruginosa and S. Typhimurium. The biofilm inhibitory activity of Ta-01 ranged from 40% at 9.38 mg/L to 60% at 150 mg/L against S. Typhimurium, and from 15% at 9.38 mg/L to more than 90% at 150 mg/L against P. aeruginosa. On the contrary, the galloquinic acid derivative, Ta-04 exhibited preferential activity against S. aureus. While the antibiofilm activity of Ta-04 against S. Typhimurium and E. coli was dose dependent (reaching a maximum of 50% biofilm inhibition against both bacteria at a concentration of 150 mg/L), the antibiofilm activity of Ta-04 against S. aureus was not dose dependent, with more than 70% biofilm inhibition at the three assayed concentrations.With respect to the hydrolysable tannins, tannic acid, Vv against S. Typhimurium and the hydrolysable tannic acid Ta-01 against E. coli at the highest concentration of compound . If there were no differences in the maximum activity, the activity at lower concentrations was also evaluated, thus allowing to determine if a derivatization was able to obtain the same effect as the unmodified tannin, but at a lower concentration.C3NMe3Cl-0.1 reduce the antibiofilm activity of the tannins, while some of them can shift the activity spectrum toward preferential activity against Gram-positive or Gram-negative bacteria. More importantly, it must be noted that, in general, the chemical derivatizations increased the activity against planktonic bacteria and decreased the activity against bacterial biofilms of the unmodified tannins, thus decreasing the biofilm specificity of the assayed tannins  already modified the antibiofilm effect of the assayed tannins, a phenomenon that could be attributed to removal of impurities that affect the antibiofilm effect of the tannins. It can be seen that Blank-W conditions increased the maximum antibiofilm effect of 3NMe3Cl, was different depending on the equivalents of chemical substitution.Regarding actual substitutions, it can be seen that the effect of a derivatization with positive charge introducing ammonium groups, i.e., C3NMe3Cl-0.5, the high equivalent derivatization, decreased the maximum antibiofilm effect of condensed tannins against S. aureus but did not affect the anti-staphylococcal effect of hydrolysable tannins. However, C3NMe3Cl-0.5 derivatization had a tannin-dependent effect against Gram-negative bacteria. It did not affect the antibiofilm effect of monomeric Vv-20 against any Gram-negative bacteria but increased the maximum antibiofilm effect of low oligomeric Vv against Salmonella Typhimurium while decreasing the maximum antibiofilm effect of Vv against P. aeruginosa. It increased the effect of high oligomeric Am against all the Gram-negative bacteria at 79.69 mg/L without significantly changing the maximum effect compared to the unmodified tannin. It increased the maximum effect of tannic acid Ta-01 against E. coli and Salmonella Typhimurium but decreased the effect against P. aeruginosa at 79.69 mg/L and it significantly decreased the maximum antibiofilm effect of galloquinic acid derivative Ta-04 against Salmonella Typhimurium. On the other hand, C3NMe3Cl-0.1, the low equivalent derivatization, reduced the maximum effect of all tannins  against Gram-negative bacteria without affecting the antibiofilm effect against S. aureus.On the one hand, C3NMe3Cl, there were no big differences between different levels of derivatizations with acidifying motif CH3COOH, i.e., CH3COOH-0.1 and CH3COOH-0.5, in terms of their impact on the activities compared to the unmodified tannins. For S. aureus, neither derivatization level affected the antibiofilm effect exerted by larger tannins Am, Ta-01, and Ta-04, comparable to the C3NMe3Cl-0.1 derivatization, while CH3COOH-0.1 increased the maximum effect of low oligomeric Vv. CH3COOH-0.5 had a similar effect on essentially monomeric Vv-20. For the Gram-negative bacteria, both derivatizations equally decreased the maximum antibiofilm effect of condensed tannins Vv-20 and Am, as well as for hydrolysable tannin Ta-04 against S. Typhimurium, but only CH3COOH-0.1 significantly decreased the maximum antibiofilm effect against S. Typhimurium of Vv. While both derivatization levels drastically decreased the maximum antibiofilm effect of condensed tannins against P. aeruginosa, only CH3COOH-0.1 decreased the maximum antibiofilm effect of tannic acid Ta-01. Neither derivatization level significantly affected the antibiofilm effect of hydrolysable Ta-04. This effect was similar regarding E. coli because neither derivatization level affected the maximum antibiofilm effect of hydrolysable tannins, while CH3COOH-0.1 decreased the maximum antibiofilm effect of all condensed tannins. CH3COOH-0.5 only significantly decreased the maximum antibiofilm effect of Vv-20 against E. coli.Contrary to C500, it can be seen that PEG500-0.05 in general did not affect the antibiofilm effect of the assayed tannins. The only exceptions are the increase in the maximum antibiofilm effect of low oligomeric Vv against Salmonella Typhimurium and the decrease in the antibiofilm effect of monomeric Vv-20 against S. aureus at 79.69 mg/L without changing the maximum antibiofilm effect. It has to be taken in account that, due to technical issues, it was not possible to analyze the effect of polymerizing PEG500-0.05 derivatization on Ta-04. Polymerizing PEG500-0.05 derivatization proofed difficult to analyze in terms of loading for Ta-04 . HL would like to additionally thank the MIUR for the Grant “Dipartimento di Eccellenza 2018-2022” to the Department of Earth and Environmental Sciences of the University of Milano-Bicocca. CC acknowledges the Ca’Foscari FPI 2019 funding.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pseudomonas aeruginosa and Salmonella typhimurium in complex samples by using wheat germ agglutinin (WGA)-modified magnetic quantum dots (Mag@QDs) as a universal detection nanoprobe. The Mag@QDs-WGA tag with a 200 nm Fe3O4 core and multiple QD-formed shell was introduced into the LFA biosensor for the universal capture of the two target bacteria and provided the dual amplification effect of fluorescence enhancement and magnetic enrichment for ultra-sensitivity detection. Meanwhile, two antibacterial antibodies were separately sprayed onto the two test lines of the LFA strip to ensure the specific identification of P. aeruginosa and S. typhimurium through one test. The proposed LFA exhibited excellent analytical performance, including high capture rate (>80%) to the target pathogens, low detection limit (<30 cells/mL), short testing time (<35 min), and good reproducibility (relative standard deviation < 10.4%). Given these merits, the Mag@QDs-WGA-based LFA has a great potential for the on-site and real-time diagnosis of bacterial samples.Point-of-care testing methods for the rapid and sensitive screening of pathogenic bacteria are urgently needed because of the high number of outbreaks of microbial infections and foodborne diseases. In this study, we developed a highly sensitive and multiplex lateral flow assay (LFA) for the simultaneous detection of  Pseudomonas aeruginosa (P. aeruginosa) and Salmonella typhimurium (S. typhimurium) are two important Gram-negative pathogens widely distributed in natural and hospital environments and responsible for numerous hospitalizations [P. aeruginosa and S. typhimurium in complex environments is key to guiding timely treatment and saving lives. Early and accurate identification of these bacteria is key to guiding timely treatment and saving lives. Commonly used techniques to detect these pathogens are bacterial isolation and culture methods, polymerase chain reaction (PCR), mass spectrometry, and next-generation sequencing [Common pathogenic bacteria pose major threats to human life and food safety because they can cause many serious infectious diseases, including pneumonia, urinary infections, diarrhea, sepsis, and bacillary dysentery ,2,3. Theizations ,7. Thus,quencing ,9,10. HoP. aeruginosa and S. typhimurium simultaneously remains to be developed.Lateral flow assay (LFA), which combines the virtues of chromatographic separation and immune recognition, has become a popular POCT method in biochemical analysis ,12,13. TP. aeruginosa and S. typhimurium and introduced it into the LFA biosensor to enable the ultrasensitive and quantitative analyses of the two target bacteria in a single test.Wheat germ agglutinin (WGA) is a well-studied phytolectin that can specifically recognize and bind to certain carbohydrates (mainly N-acetylglucosamine and its derivatives) on the cell wall of most bacteria . CompareP. aeruginosa and S. typhimurium from complex samples, which allows the stable detection of bacteria through the LFA strip. Secondly, the high-performance Mag@QDs nanoprobe is fabricated by coating two layers of carboxylated QDs (CdSe@ZnS-COOH) onto 200 nm Fe3O4 MNPs, which provides the dual amplification effect of multiple QDs and magnetic enrichment, ensuring the high sensitivity of the proposed assay. Thirdly, the Mag@QDs-WGA-based LFA allows the on-site and simultaneous detection of two target pathogens in 35 min with a low detection limit  by constructing two antibody-loaded test lines. To the best of our knowledge, this study is the first to report the direct and multiplex detection of P. aeruginosa and S. typhimurium via LFA. In addition, the proposed biosensor shows good stability, specificity, and accuracy for the detection of complex bacterial samples (containing food and environment samples). This study suggests that the Mag@QDs-WGA-based LFA has a great potential for POCT applications.The proposed Mag@QDs-WGA-based LFA has three great innovations compared with previous LFA biosensors for pathogens. Firstly, the Mag@QDs-WGA acts as the universal capture platform for the rapid magnetic enrichment of The materials, chemicals, and apparatures used in this work are described in Supporting Information S1.1 and S1.2.3O4 nanobeads (MB) 200 nm in diameter were fabricated via a classical solvothermal reaction [3O4@QD-shell nanobeads (Fe3O4@QDs) were magnetically collected, rinsed with deionized water, and then dispersed in 5 mL of ethanol. Afterward, the prepared Fe3O4@QDs were successively reacted with PEI and CdSe/ZnS-MPA QDs, and Fe3O4@double QD-shell nanobeads (Mag@QDs) were obtained through PEI-mediated self-assembly. Finally, the obtained Mag@QDs were dried under vacuum at 60 °C, resuspended in 10 mL of absolute ethanol solution, and then stored away from light for further use.The synthesis of the Mag@QDs nanobeads (Mag@QDs) is shown in reaction . Then, 5reaction . After mWGA was directly modified onto the Mag@QDs through EDC/NHS chemical coupling. In brief, 1 mL of Mag@QDs (1 mg/mL) was added into a 2 mL EP tube. Then, the Mag@QDs were magnetically separated from the solution and resuspended in 500 µL of MES solution . Subsequently, 5 µL of EDC (0.1 M) and 10 µL of NHS (0.1 M) were added into the mixture and sonicated for 15 min. The activated Mag@QDs were magnetically enriched and resuspended in 200 µL of PBS buffer . Afterward, 20 µL of aqueous WGA (1 mg/mL) was added to the mixture and incubated for 2 h to form Mag@QDs-WGA. After collecting the production with an external magnetic field, 100 µL of glycine solution (0.75 mg/mL) was added and shaken for another 1 h to block the excess carboxyl group. Finally, the obtained Mag@QDs-WGA was washed twice and stored in 200 µL of PBS buffer  for further use.P. aeruginosa (1 mg/mL) and S. typhimurium (0.8 mg/mL) were coated on the NC membrane by using an XYZ spraying machine  to form test line 1 (T1) and test line 2 (T2), respectively. The volume of bacterial detection antibody was 20 µL, and the spray rate was set as 1 µL/cm. Next, the antibody-coated NC membrane was dried for 3 h in a 37 °C environment and assembled on the plastic back card of the LFA. Finally, the LFA card consisting of a sample pad, an NC membrane, and an absorption pad were cut into 3 mm-wide LFA strips for subsequent use.Antibodies to P. aeruginosa and S. typhimurium were inoculated on agar plates containing 5% sheep blood and grown at 37 °C, 5% CO2 for 12 h. Then, about 20 colonies were picked from the plate and added into 1 mL of PBS  as the original bacterial solution. After diluting the bacterial solution 1 × 105–1 × 107 times, 100 µL of the solution was spread on a blood agar plate and incubated as described before. Based on the counting results of colony forming units on the plate, the concentration of the original bacterial solution was calculated and adjusted to the detection concentration.The plate culture method was used to quantify the bacteria . The pro3 cells/mL). After 20 min of shaking, the Mag@QDs-WGA-bacterial mixture was magnetically enriched. Then, 100 µL of supernatant liquid was placed on the blood agar plate. After 12 h of incubation, the uncaptured bacteria were counted, and the capture rate was calculated.Then, we investigated the bacterial capture rate of Mag@QDs-WGA in the complex solution. In brief, 4 µL of Mag@QDs-WGA (5 mg/mL) was added into 1 mL of the bacterial sample  and 4 µL of Mag@QDs-WGA were added into 1 mL of sample solution. After shaking for 15 min, the tube containing mixture was placed on an external magnet. The Mag@QDs-WGA-bacteria complexes in the sample were rapidly enriched to the bottom of EP tube under the action of magnetic field (<2 min), and then the supernatant was removed by a pipette. Subsequently, the complexes were resuspended in 100 µL of running buffer  and loaded onto the sample pad of the LFA strip. After the 15 min of chromatographic reaction, the test strip was inserted into a portable fluorescence reader  to simultaneously read the fluorescence signals of two test lines. As shown in Different real samples  containing 0–10S. typhimurium and P. aeruginosa in complex samples. The nanoprobe Mag@QDs-WGA is a key factor affecting the performance of fluorescent LFA. It consists of three components: (i) a 200 nm Fe3O4 core to ensure strong magnetic responsiveness for rapid enrichment, (ii) two layers of QD-formed shell to provide strong fluorescence intensity and large surface area, and (iii) surface-modified WGA molecules to recognize a broad spectrum of pathogens and effectively bind S. typhimurium and P. aeruginosa in complex samples.P. aeruginosa and S. typhimurium. The operation principle of the Mag@QDs-WGA-based fluorescence LFA can be divided into two parts. Firstly, the Mag@QDs-WGA universal tags were used to capture a broad spectrum of bacteria in solution and separate them via a magnetic field  containing 0.2 g/mL of the three sugars as test samples and Mag@QDs-WGA was added to capture bacteria. As shown in WGA is a protein specific for N-acetylglucosamine and its derivatives. Thus, the interference of common monosaccharides on WGA required further investigation. We selected three common sugars  as interfering substances. To the best of our knowledge, the content of the three sugars in common food products is lower than 0.2 g/mL. Therefore, we prepared bacterial solutions  and S. typhimurium (R2 = 0.999) ensured the reliability of Mag@QDs-WGA-LFA for quantitative bacterial detection. The precise concentrations of the prepared bacterial samples were determined using plate colony counting. As displayed in The ability of Mag@QDs-WGA-LFA for the ultrasensitive and quantitative detection of both pathogens was validated under optimized conditions. A series of samples containing 10re drawn f,g. AccoP. aeruginosa and S. typhimurium are large (0.5 µm wide and 1–2 µm long), whereas the Mag@QDs-WGA nanoprobe is small (approximately 200 nm diameter). Thus, one separate bacterium can usually bind to multiple nanoprobes. Whether the number of probes we add in can capture all the bacteria in the sample can directly affect the quantitative ability of the dual-channel detection. In the present study, we verified the sensitivity of the single-channel detection of bacteria and compared it with the results of dual-channel detection (6 cells/mL) of P. aeruginosa and S. typhimurium were prepared and detected by Mag@QDs-WGA-LFA. The fluorescent image of the test strips showed that the target bacteria in the solution could produce a red band on the corresponding detection line and had no significant effect on another test line. The visual detection limit of the samples containing only P. aeruginosa or S. typhimurium was 50 cells/mL. The test lines of the two groups were detected using a portable fluorescence instrument, and the fluorescent signals are shown in P. aeruginosa and S. typhimurium were 23 and 25 cells/mL, respectively. The results of the single-channel test  under UV light showed that the minimum visualized concentration of P. aeruginosa was 50 cells/mL. P. aeruginosa was 22 cells/mL, which was close to the detection limit of Mag@QDs-WGA-LFA. The results indicated that WGA can reach the sensitivity of antibodies and can be employed as a low-cost antibody substitute in LFA for bacterial detection.Then, the performance of the LFA based on the WGA proposed in this study was compared with that of the traditional dual-antibodies sandwich-type method. The probe in the LFA of the traditional dual-antibody sandwich method was immuno-Mag@QDs, which were Mag@QDs directly modified with anti-6, 104, and 102 cells/mL of bacterial samples by using Mag@QDs-WGA-LFA. The fluorescence signals of all LFA band test areas were relatively uniform, and their relative standard deviation (RSD) values ranged from 3.96% to 9.46%, indicating the high reproducibility and stability of the proposed method. The spraying antibody ensures the specificity of Mag@QDs-WGA-LFA; thus, it also needed to be verified. We prepared six samples of non-target bacteria, including Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Campylobacter jejuni, Shigella flexneri, and Vibrio parahaemolyticus, and the same concentration (106 cells/mL) was detected using Mag@QDs-WGA-LFA to evaluate the specificity of the proposed strategy. As shown in P. aeruginosa, S. typhimurium, and P. aeruginosa/S. typhimurium mixture) produced strong fluorescence intensity on the corresponding test line. The results showed that the method has good specificity to target bacteria and strong anti-interference ability to non-target bacteria.Reproducibility and specificity are important indexes to evaluate Mag@QDs-WGA-LFA. S. typhimurium and P. aeruginosa are mainly transmitted through contaminated food or water sources. Two common food samples  and one environmental sample (river water) were selected as real samples and added with 1 × 106, 1 × 104, and 1 × 102 cells/mL of pathogens (P. aeruginosa and S. typhimurium), respectively. Real sample analysis was carried out following the established scheme described in We then evaluated the detection performance of Mag@QDs-WGA-LFA in real food and environmental samples because P. aeruginosa and S. typhimurium using WGA-modified Mag@QDs as a broad-spectrum capture tool and fluorescent nanoprobe. The WGA-functionalized Mag@QDs nanoprobe showed great advantages over traditional antibody-modified signal tags, including broad-spectrum bacterial recognition, high affinity to bacteria, magnetic enrichment ability, superior fluorescence property, and low cost. By introducing the Mag@QDs-WGA into the LFA system, the proposed biosensor can simultaneously detect P. aeruginosa and S. typhimurium with LODs of 25 and 28 cells/mL, respectively. Moreover, the established Mag@QDs-WGA-LFA showed high reliability, stability, and accuracy when applied to detect complex bacterial samples. In theory, the detection strategy can be easily applied to other pathogenic bacteria by changing the specific antibodies on the test lines of the LFA strip because of the broad-spectrum recognition ability of WGA. These results indicate that the proposed Mag@QDs-WGA-LFA has great potential to be developed into a powerful tool for the rapid screening of bacteria in field samples.In this study, we reported a two-channel fluorescence LFA biosensor for the simultaneous detection of


In addition, TEMPO-CNF can be used as an emulsion stabilizer for lemongrass essential oil loaded oil-in-water emulsion. This study is among the first to report that TEMPO-CNF improved the freeze-thaw stability of oil-in-water emulsions stabilized by small molecular weight surfactants .Agricultural byproducts such as corn stover are widely available sources for preparation of nanocellulose, which is an emerging green chemical with versatile applications. In this study, corn stover-derived nanocellulose was prepared via bleaching, alkaline treatment, 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) oxidation, and ultrasonication. The as-prepared TEMPO-oxidized cellulose nanofibril (TEMPO-CNF) was characterized by transmission electron microscopy, UV-Vis spectrophotometry, rheometry, and zeta potential measurement. Droplet size, phase behavior, and thermodynamic stability of TEMPO-CNF stabilized oil-in-water emulsions were investigated. Results show that TEMPO-CNF with a width of 4 nm, length of 353 nm, and surface charge of 1.48 mmol/g COO According to a US Department of Energy report, more than 75 million dry tons of corn stover are produced every year. Investigation of value-added use of corn stover can increase economic and environmental sustainability. With a cellulose content of ~45%, corn stover is a widely available cheap source for the preparation of nanocellulose. Nanocellulose is a cellulosic nanomaterial with at least one dimension less than 100 nm. Nanocellulose is biodegradable and biocompatible with negligible toxicity, and has been widely applied in fields such as pharmaceutics, packaging, textiles, and cosmetics ,3,4,5,6.Emulsions can be stabilized by low molecular weight surfactants (such as Tween 80) or high molecular weight biopolymers. For instance, small molecular weight synthetic surfactant Tweens have been widely used to stabilize emulsion products . HoweverNanocellulose has been shown to be an effective Pickering emulsion stabilizer ,20,21,22The corn stover sample was received from the Iowa State University BioCentury Research Farm  and ground into a size below 1/8 inch. All of the chemicals were purchased from VWR  or Fisher Scientific .Corn stover was used as the source material for the preparation of nanocellulose according to the methods described by Costa et al.  with modAlkaline treatment was followed by using 4 wt% NaOH with a solid–liquid ratio of 20:1 at 80 °C in a shaking water bath at 120 rpm for 2 h, followed by filtration with a 25 µm sieve and washing until the affluent was clear. Lastly, the corn stover sample was dried and ground with the above-mentioned grinder, followed by ball milling  at 100% speed for 10 min to further reduce the particle size. The sample was then passed through a 40-mesh sieve and used for TEMPO oxidation treatment.4 was added to the suspension with continuous stirring. After 3 h, the suspension was centrifuged  at 7745 g for 10 min at 4 °C. The pellet was washed with DI water, followed by centrifugation for 3 times, then TEMPO-CNF gel was obtained. The solid content of the concentrated gel was determined by measuring the moisture content of the sample. The diluted gel sample was prepared by diluting the obtained gels with DI water, followed by ultrasonication using a 500 W ultrasonicator  for 2 h in an ice bath, centrifugation at 7745 g for 10 min at 4 °C and collection of the supernatant. Centrifugation was applied to remove any impurities released from the ultrasonic probe, or to remove any un-fibrillated and partly fibrillated fractions if present.After bleaching and alkaline treatment, the corn stover sample was used to prepare TEMPO-CNF using the TEMPO/NaBr/NaClO oxidation system, according to Okita et al.  with modn = 1, 2, 3, 4.The color of the corn stover sample after each treatment was recorded by a colorimeter . The yields of the corn stover sample after each treatment were calculated by the following equations. Specifically, step yields and overall yields were both calculated.w/v)) was added to a beaker; then, the pH of the suspension was adjusted to 2.0 by addition of 0.1 M HCl to make the carboxylate groups in protonated forms. The suspension was allowed to stand for 15 min; then, 1.0 mL of 1.0 mM NaCl was added, and the suspension was stirred for 90 min before titration. Titration was performed with the addition of 0.1 M NaOH at the rate of 0.1 mL per 30 s. The conductivity value of the suspension during each addition of NaOH was recorded using an Oakton PC 700 pH meter , and the titration was continued until the pH of the suspension reached 10.5. The carboxylate content of the sample was calculated from the conductivity and pH curves. Specifically, the titration curves show the presence of a strong acid and a weak acid, which correspond to the excess of HCl and the carboxylate content, respectively. The carboxyl content was calculated using the equation below.N represents the carboxyl content (mmol/g), V1 and V0 represent the equivalent volumes (mL) of added NaOH solution, NaOHC represents the concentration (mol/L) of NaOH solution, and m represents the dried mass (g) of sample.The carboxyl content of the prepared nanocellulose was determined according to the methods described by Liu et al.  with mod−1 and a 180 s period  was performed at 37 °C. Three tests were performed for each sample and the viscosity readings versus shear rate were recorded. The dimensions of the as-prepared TEMPO-CNF were determined using transmission electron microscopy (TEM) according to the methodology described in Costa et al. . SpecifiTo prepare nanocellulose-stabilized Pickering emulsions, TEMPO-CNF suspensions at varying concentrations were added to lemongrass essential oil (EO) with magnetic stirring, followed by ultrasonication with a 500 W ultrasonicator for 5 min. The EO-loaded emulsion contained 2.5 wt% EO and certain concentrations of TEMPO-CNF . The emulsion samples were stored at room temperature (away from light) and observed for phase separation over time. Images of the TEMPO-CNF-stabilized emulsions were captured with an optical microscope  immediately after sample preparation. Specifically, 10 µL of undiluted sample (right after sample preparation) was pipetted onto a microscope slide and covered with a coverslip. Images were taken under 40× objective magnification. Droplet size of the emulsion was determined from the images using Image J 1.53t software . Emulsion samples with the presence of both TEMPO-CNF and a low molecular weight surfactant  were also prepared. The EO-loaded emulsions contained certain concentrations of lemongrass EO , certain concentrations of Tween 80 (EO/Tween 80 ratio 1:3), and with or without the presence of TEMPO-CNF (0.3 wt%). Emulsions were prepared by slowly adding Tween 80 to EO with stirring, then the aqueous phase (containing nanocellulose and water) was added with stirring, followed by ultrasonication with a 500 W ultrasonicator for 5 min.Several thermodynamic stability tests, such as centrifugation and freeze-thaw tests of emulsion samples, were performed to assess their stability. For the centrifugation test, emulsion samples were centrifuged at 10,000 rpm  for 15 min at 25 °C and observed for phase separation (if any). During the freeze-thaw test, emulsion samples were stored for 2 cycles with each cycle of storage at −20 and 25 °C for 48 h each, then observed for phase separation (if any). The appearance of the corn stover sample after each treatment is shown in According to Acetobacter xylinum bacteria (1.05 mmol/g), Tunicates of Halocynthia roretzi (0.59 mmol/g), and whole plants of Cladophora sp. (0.52 mmol/g) [In addition, it is determined from  mmol/g) . With 10 mmol/g) . At carb mmol/g) .−1 shear rate (η) as a function of TEMPO-CNF concentration (C) is shown in 10 η = a + blog10 C), which is similar to that of our previous studies [2 = 0.96. At a shear rate of 20 s−1, viscosity increases at higher TEMPO-CNF concentrations. Specifically, the viscosity of TEMPO-CNF at 3.81 wt% is 6.75 Pa·s, which is much higher than that of 0.7 wt% (0.33 Pa·s), 0.5 wt% (0.105 Pa·s), 0.3 wt% (0.013 Pa·s), and 0.1 wt% (0.003 Pa·s). Based on the fitting parameters, the viscosity of corn-stover derived TEMPO-CNF (our study) is slightly lower than that of wood-derived TEMPO-CNF (produced by USDA’s Forest Products Laboratory), as wood-derived TEMPO-CNF had fitting parameters of a = 5.45 and b = 2.42, as shown in our previous study [The viscosity of TEMPO-CNF at different concentrations is shown in  studies ,34. The us study , reflectus study .Lemongrass essential oil loaded emulsions with or without the presence of TEMPO-CNF did not show any phase separation right after sample preparation (result not shown here). However, after 24 h of storage at room temperature (absence from light), the emulsions prepared without TEMPO-CNF or with low concentrations of TEMPO-CNF (below 0.5 wt%) underwent phase separation. No phase separation was observed in emulsions stabilized by TEMPO-CNF at concentrations above 0.5 wt%. The percentages of the creaming layers in the emulsions are shown in A similar phenomenon of concentration-dependent emulsion stability against creaming was also observed for Pickering emulsions stabilized by other biopolymers such as starch nanocrystals , chitin 32), and, in general, smaller d32 corresponds to better emulsification performance [32) of hexadecane-in-water emulsions decreased as the bacterial cellulose nanocrystal concentration increased until it reached a minimum value [formance . It was um value . The effum value  and Yangum value . For insum value .It was reported that solid particles can irreversibly adsorb on droplet surface. At low particle concentrations, emulsion droplets are only partially covered by the particles, thus, the system is not stable. However, at higher particle concentrations, excess particles form a network structure surrounding droplets and enhance viscosity, thus improving the system stability . As showIt was also reported that TEMPO-CNF can form nanosized emulsion droplets. Specifically, emulsion droplets with a size as low as 350 nm were achieved by cotton TEMPO-CNF . TEMPO-CEmulsions stabilized by TEMPO-CNF (0.3 wt%) and a low molecular weight surfactant  were also studied. Thermodynamic stability tests were performed, and comparisons were made among emulsions stabilized by Tween 80 with or without the presence of TEMPO-CNF. Results are shown in The inability of Tween 80 to stabilize the oil-in-water emulsions against the freeze-thaw process was also reported by Ariyaprakai and Tananuwong . SpecifiPolymers with ice recrystallization inhibition activity have been shown to enhance the freeze-thaw stability of materials . TEMPO-CTo the best of our knowledge, the current work is the first to report the ability of TEMPO-CNF to improve the freeze-thaw stability of Tween 80-stabilized oil-in-water emulsion. Based on the findings of this study, it was postulated that both TEMPO-CNF and Tween 80 adsorbed on the oil-water interface. Synergistic effects may occur between TEMPO-CNF and Tween 80 for stabilization of oil-in-water emulsions. Similarly, synergistic effects have been reported between nanocellulose and saponin  . In addiAccording to Dickinson , the pre-. At 0.1 wt%, the colloidal suspension of TEMPO-CNF had a light transmittance of ~100% at a wavelength of 400–1100 nm, indicating that the suspension is transparent. At higher concentrations, the colloidal suspension of the prepared TEMPO-CNF had higher viscosity. Lemongrass essential oil loaded emulsions stabilized by TEMPO-CNF at concentrations above 0.5 wt% did not undergo any phase separation after 24 h of storage at room temperature. Decreased emulsion droplet size was observed in emulsions stabilized by higher TEMPO-CNF concentrations. Specifically, an average droplet size of 1.67 µm was observed in emulsions stabilized by 0.7 wt% TEMPO-CNF. The presence of TEMPO-CNF did not affect the stability of Tween 80 stabilized oil-in-water emulsions against centrifugation, that is, both types of emulsions did not form phase separation after centrifugation. Furthermore, to the best of our knowledge, this is the first study to report that TEMPO-CNF possesses the capability to improve the freeze-thaw stability of Tween 80 stabilized oil-in-water emulsions. Findings from this study lay a foundation for further application of nanocellulose as an emulsion stabilizer against environmental stresses. The developed emulsion systems are also consistent with growing interest in using natural substances to make stable emulsion products.In this work, corn stover-derived TEMPO-CNF gel with a solid content of ~3.8 wt% was prepared, characterized, and studied as an emulsion stabilizer. The as-prepared TEMPO-CNF had a width of 4 nm, length of 353 nm, and surface charge of 1.48 mmol/g COO


IntroductionUretero-ureterostomy combined with unilateral nephrostomy is a rarely performed urinary diversion following radical cystectomy for muscle-invasive bladder cancer. The aim of this study is to assess the efficacy and safety of the procedure.Materials and methodsPatients with muscle-invasive bladder cancer and poor performance status were enrolled in this retrospective, observational, single-centre study, carried out between December 2018 and November 2020. The patient's renal function was regularly assessed with serum creatinine and estimated glomerular filtration rate (eGFR). Evaluation of peri- and postoperative complications was performed based on clinical, laboratory, endoscopic, ultrasound and other radiological studies findings. The patient’s status was assessed for 12 months.ResultsThirty-six patients with a mean age of 77.4±8.6 years were enrolled in the study. 86.11% of patients had an American Society of Anesthesiologists Score ≥3 and 91.66% had an age-adjusted Charlson comorbidity index of ≥6. Slight deterioration of renal function, although not statistically significant, was observed. 36.11% of the patients developed permanent unilateral pelvic dilatation. Acute pyelonephritis, urosepsis, pyonephrosis and anastomotic leak were observed in 22.22%, 8.33%, 5.55% and 5.55% of patients, respectively; all were treated either conservatively and/or with minimally invasive procedures  without any need for open surgical revision.ConclusionsUreteroureterostomy combined with unilateral nephrostomy is a safe and effective method of urinary diversion following radical cystectomy for muscle-invasive bladder cancer with easily manageable complications. Bladder cancer (BC), which is the 10th most common newly diagnosed malignancy, ranks 13th in deaths worldwide .In patients with muscle invasive, as well as in those with BCG-refractory non-muscle-invasive BC, radical cystectomy is recommended ,4, with The first reference to uretero-ureterostomy was made in 1961 by Arnold et al. in the USA . While tPatients with stage T2-4b N0/N1 M0/M muscle-invasive BC, without osseous metastases, being at high surgical or anaesthesiologic risk and/or low life expectancy scheduled for radical cystectomy were included in the present study, which took place in the Department of Urology in General Hospital of Hagen in Germany. Exclusion criteria were a solitary kidney , bone metastases, haemodialysis, stricture of the upper third of the ureter, bilateral staghorn calculi, retroperitoneal fibrosis and previous ureterolysis. All surgical procedures were performed under general anaesthesia by two experienced urological surgeons. Ceftriaxone was administered intravenously as chemoprophylaxis. Preoperatively, an ultrasound-guided percutaneous nephrostomy tube 14-16 F was placed. After excision of the bladder, both ureters were developed cranially up to the level of the common iliac artery, where one ureter  was transferred contralaterally, through a tunnel under the root of the mesentery, to be anastomosed with the other in an end-to-end fashion, using 5-0 absorbable interrupted sutures. The anastomosis was carried out over a 7F 40cm JJ catheter, which was inserted over a hydrophilic guidewire in both renal pelves. After the JJ stent is removed percutaneously under sedoanalgesia in the sixth postoperative week under pre-interventional, targeted antibiotic prophylaxis via the existing nephrostomy tube channel using a nephroscope, the nephrostomy tube is changed.Absolute success was defined as the survival of the patient without anastomosis complications requiring open surgical revision with the non-fatal complication of anastomosis and eGFR reduction of less than 25%. Qualified success was defined as the survival of the patient without anastomosis complications requiring open surgical revision with the non-fatal complication of anastomosis.Follow-up included scheduled visits to Clinic after six weeks and at the third, sixth, ninth and 12th postoperative months. Clinical examination, laboratory studies and sonographic and radiological studies were carried out during the visits. Renal function was assessed using serum creatinine and estimated glomerular filtration rate (eGFR). The ureteric stent was removed at the sixth postoperative week, whereas replacement of the nephrostomy tube was carried out every six weeks, under fluoroscopic guidance.All patients signed an informed consent form for surgery. The tenets of the Declaration of Helsinki were fully respected. The study was approved by the local Institutional Review Board Committee.Statistical analysis was carried out with IBM Statistical Package for the Social Sciences (SPSS) statistics version 22. We were able to examine with an independent sample t-test. The normality of the data was assessed using a histogram as a graphical method. Because the examined parameters did not follow a normal distribution, we used also the nonparametric Wilcoxon sign rank test. Statistical significance was set at 5% (p=0.05). In addition, we performed a Kaplan Meier survival analysis. An a-priori power analysis using G*Power 3  to test Thirty-six patients  with a mean age of 77.4±8.6 years were included in the study. Thirty-one patients (86.11%) had an American Society of Anaesthesiologists Score ≥3 (ASA-score ≥ 3), whereas 33 (91.66%) had an age-adjusted Charlson comorbidity index ≥ 6. Patients were followed for a mean of 14.73±8.2 months. We herein present our data at the 12-month follow-up. Two patients were excluded from subsequent follow-up in the third and ninth month because of the insertion of a nephrostomy tube in the contralateral kidney due to complications of the anastomosis. The previous history of urothelial cancer, smoking, chronic exposure to hazardous substances in the workplace and pelvic radiation were risk factors for BC in 13, 29, seven and six patients, respectively. Patients’ demographics and type of surgery for BC are presented in Table Renal function was found to slightly deteriorate over the follow up period, although not significantly. Overall, mean serum creatinine and eGFR changed from 1.20±0.39 mg/dL and 58.33±18.88 mL/min preoperatively to 1.25±0.39 mg/dL (p=0.30) and 56.67±21.05 mL/min (p=0.38) after six months and 1.41±0.54 mg/dL (p=0.051) and 52.10±19.64 mL/min (p=0.13) after 12 months  and mean eGFR  levels at the 12-month follow-up. Renal function was also stable in patients with postoperative pelvic dilatation. Oehler et al. reported uretero-ureterostomy with definite nephrostomy to have no impact on renal function in eight patients . SimilarAcute pyelonephritis was diagnosed in eight patients (22.22%) postoperatively, although all patients were found to have a positive culture in urine collected from their nephrostomy tube. This incidence is in accordance with the reported in the literature for uretero-cutaneostomy and transuretero‑cutaneostomy 2.27%-42.30%) -27. In s%-42.30% Infected hydronephrosis was diagnosed in two patients. The first case was a random finding and the second patient suffered acute pyelonephritis and belongs to the above-mentioned eight patients of our cohort Table . The conThe overall rate of postoperative complications was low in our series. Although two patients died of complications related to surgery, these deaths were not associated with the uretero-ureterostomy per se. Furthermore, no skin strictures were noticed, as opposed to a 3.7%-75.9% stoma stricture rate occurring in patients with uretero-cutaneostomy ,24,25,27Leakage from the anastomosis was noticed in two cases; the first was considered a failure in the technique of the anastomosis, as it occurred in the early postoperative period, whereas the other resulted from injury of the anastomosis during a subsequent sigmoidectomy for the treatment of obstructive ileus. Both cases were successfully treated with insertion of a second nephrostomy in the contralateral kidney. Furthermore, a ureteral JJ-stent was percutaneously inserted under sedoanalgesia in the above mentioned patient with the intraoperatively injured uretero-ureterostomy; both nephrostomy tubes were also left in place in this case. Khalilullah et al. reported stricture and leakage of the ureteric anastomosis in 4.5% of patients , slightlComplications, including tube dislodgement, inability to remove the JJ-stent and anastomotic insufficiency were successfully treated by minimally invasive replacement of a nephrostomy under sedoanalgesia. No patient required a second open surgical procedure to treat postoperative complications. One patient refused ipsilateral exchange of the nephrostomy because of the discomfort he experienced in his preferred side-sleeping position. Thus, the placement of a nephrostomy in the contralateral kidney after tube obstruction was not considered to be a complication. The above-mentioned complications are considered as minor in comparison to the mentioned complications of the studies of Khalilullah and Wandschneider. Khalilullah et al. noted that 4.5% of their cohort underwent a re-operation due to a leaking ureteric anastomosis and 22.7% required JJ-stenting to treat stomal stricture . MoreoveLimitations of our study include the small number of patients and the relatively short follow-up. In addition, this is a single-arm study and the comparison of the renal function with other types of urinary diversion could not be done. Further studies are needed to evaluate the long-term efficacy and safety of uretero-ureterostomy in combination with unilateral nephrostomy following radical cystectomy.This study demonstrated that uretero-ureterostomy combined with unilateral nephrostomy following radical cystectomy is a safe and effective method of urinary diversion for elderly patients with muscle-invasive BC who have a poor performance status. While larger, longer-term studies are needed to confirm these results, the absence of either severe surgical complications requiring re-operations or diversion-related deaths, as well as the satisfactory preservation of renal function indicate this type of urinary diversion to serve as a reliable alternative in this, difficult to treat, patient population.


As the most widely used neonicotinoid insecticide, it is of great significance to explore the immunoreagents and immunoassays for imidacloprid (IMI) residue. In immunoassays, specific peptide ligands, such as peptidomimetic and anti-immunocomplex peptides, are regarded as promising substitutes for chemical haptens. In the present work, we identified thirty sequences of peptidomimetics and two sequences of anti-immunocomplex peptides for IMI from three phage pVIII display cyclic peptide libraries, in which the anti-immunocomplex peptides are the first reported noncompetitive reagents for IMI. The peptidomimetic 1-9-H and anti-immunocomplex peptide 2-1-H that showed the best sensitivity were utilized to develop competitive and noncompetitive phage enzyme-linked immunosorbent assays (P-ELISAs), with a half inhibition concentration of 0.55 ng/mL for competitive P-ELISA and a half-saturation concentration of 0.35 ng/mL for noncompetitive P-ELISA. The anti-immunocomplex peptide was demonstrated to greatly improve the specificity compared with competitive P-ELISA. In addition, the accuracy of proposed P-ELISAs was confirmed by recovery analysis and HPLC verification in agricultural and environmental samples. These results show that the peptide ligands identified from phage display library can replace chemical haptens in the immunoassays of IMI with the satisfactory performance. Immunoassay plays an increasingly important role in pesticide residue detection. Although the precision and reliability of chromatography-based methods are apparent, the advantages of immunoassay, such as its low-cost, simple operation and portable detection device, make it the preferable approach for screening large samples and for on-site detection. The establishment of immunoassay is realized by the recognition between immunorecognition reagents (antibody and antigen) ,2. The cIn recent years, the specific peptide screened from the phage display peptide library has shown the potential to replace chemical hapten as the next-generation immunoreagent in pesticide residue immunoassay . The pep−1 to 10 mg kg−1 (GB2763-2021). The EU has regulated the MRLs of imidacloprid in 381 kinds of food with the range of 0.01 mg kg−1 to 15 mg kg−1 (EU 2021/1881). The U.S. Environmental Protection Agency has regulated the MRLs of imidacloprid in 134 kinds of food with the range of 0.02 mg kg−1 to 240 mg kg−1. Even though IMI has been banned from outdoor use in EU, it is still necessary to monitor IMIs concentration in the environment. So, it is desirable to develop the analytical method for imidacloprid residue. Currently, dozens of immunoassays for IMI residue have been reported, most of which are competitive immunoassays based on chemical haptens  × 100%.The specificities of P-ELISAs, defined as the cross-reactivities (CRs) with other analogs of IMI, were calculated as follows: CR = [ICThe accuracies of proposed P-ELISAs for IMI detection in various agricultural  and environmental samples (paddy water and soil) were evaluated. The samples were collected from Nanjing, China. The agricultural samples were chopped and homogenized. The paddy water samples were filtered, while the soil samples were air-dried, crushed, and passed through a 2 mm sieve. All of the samples were stored at −20 °C. The samples were confirmed not to be contaminated with IMI by HPLC.The samples were spiked with various concentrations of IMI standard solutions and stood overnight. Before the analysis of the spiked samples, their matrix effect on P-ELISAs were firstly investigated by preparing the dose-response curves in series diluted blank extracts, then compared with the PBS-based standard curve to determine the dilution factors. After dilution, the paddy water was directly detected without further processing. For other solid samples, 5 g samples were added to a 50 mL centrifuge tube, then extracted by 10 mL optimized PBS containing 45% methanol. The tube was vortexed at 300 rpm for 5 min, sonicated for 15 min, then vortexed for another 5 min. After centrifuging at 4000 rpm for 5 min, the extracts were collected, and the extracts of cabbage, pear, potato, and orange were adjusted to 15 mL by PBS. The extracts of spiked samples were tested by P-ELISAs after proper dilutions.IMI standard solutions with unknown concentrations were spiked into pear and soil samples, respectively. For P-ELISAs, the samples were processed and analyzed as above. For HPLC, the samples were processed and analyzed as previously described .lac promoter/operator. Such a display strategy may increase the apparent affinity and weaken the growth bias among different phages, thus serving as a footstone for the blended panning strategy using mixed libraries [The peptide libraries used in this study are displayed on phage pVIII in high-density, whose expressions are controlled by the ibraries .The phage titer of each panning is shown in 450 ≈ 1 were chosen to develop dose-response curve for competitive P-ELISA (50 and the highest maximum OD450 (ODmax)/IC50 were selected for subsequent experiments, while the anti-immunocomplex peptides 2-1-H were selected.The sensitivities of seven peptidomimetics  that showed enhanced signal differences in the presence of IMI, and all anti-immunocomplex peptides , were evaluated by P-ELISAs. The optimal concentration of mAb and input quantities of phage-displayed peptides were firstly determined. The dosages of mAb 3D11B12E5 and phage-displayed peptides that generated the OD P-ELISA c. The do P-ELISA d. The pe50 (or SC50) and the highest ODmax/IC50 (or ODmax/SC50) were considered as the optimums. As shown in + and pH 6.0 was chosen to develop competitive P-ELISA, and the PBS buffer containing 0.07 M Na+ and pH 8.0 was chosen to develop noncompetitive P-ELISA. Reaction between the antibody and phage display peptides was significantly disturbed when the methanol concentrations in the buffer were higher than 2.5%  of 0.30 ng/mL and detection range (IC10–IC90) of 0.30–1.00 ng/mL, while the SC50 of noncompetitive P-ELISA was 0.35 ng/mL, with the LOD of 0.15 ng/mL and detection range (SC10–SC90) of 0.15–0.80 ng/mL. The noncompetitive P-ELISA showed slightly improved sensitivity than competitive P-ELISA. We have summarized the reported sensitivities of the basic immunodetection, that is ELISA, for the detection of IMI (50 = 0.067 ng/mL). The HRP labelled anti-M13 antibody used in this work is targeted at phage pVIII, whose binding may be hampered by the phage pVIII displayed peptides, thus causing a lower signal amplification effect and leading to the poor sensitivity of P-ELISAs. It is worth mentioning that isolated peptides here are cyclic peptides, which may mean better stability. Moreover, the noncompetitive immunoreagent for imidacloprid is reported for the first time, which can derive a series of noncompetitive immunoassays for imidacloprid with more intuitive detection results. It has advantages in the development of visual detection.The dose-response curves of two P-ELISAs are shown in n of IMI . Except e IC10–IC0 of 0.30e IC10–IC0 of 0.30The specificity of P-ELISAs, defined as CR, is shown in The matrix effect that would cause the false result has to be solved before testing actual samples. Owing to the high sensitivity of immunoassays, the matrix effects of extracts can simply be eliminated by the dilution method. To evaluate their influence, series diluted matrix extracts were used to replace the optimal buffer and run the dose-response curves of P-ELISAs. When the dilution fold produces a similar curve to the standard curve, the matrix effect is considered to be eliminated. As shown in After the elimination of matrix effects, the IMI concentrations in the spiked samples were determined . The ave2 = 0.9739) for pear and y = 0.98x + 5.07 (R2 = 0.9837) for soil, while y = 0.86x + 29.82 (R2 = 0.9629) for pear and y = 0.94x + 8.13 (R2 = 0.9964) for soil between noncompetitive P-ELISA and HPLC. The slope and R2 of the equations are close to 1, which indicates a good correlation between HPLC and the immunoassays.As a gold standard for pesticide residue detection, HPLC was used to further verify the accuracy of the proposed P-ELISAs. The blind samples of pear and soil were simultaneously detected by HPLC and the P-ELISAs. The results are shown in 50 and LOD of competitive P-ELISA were 0.55 ng/mL and 0.3 ng/mL, compared with 0.35 ng/mL and 0.15 ng/mL for noncompetitive P-ELISA. The sensitivities of P-ELISAs were better than most reported immunoassays for IMI, which can be further improved by collaborating with other tracers. Moreover, the two-site recognition pattern of anti-immune complex peptides in noncompetitive P-ELISA greatly improved the specificity compared with competitive P-ELISA. As confirmed by the recoveries analysis and HPLC verification, the proposed P-ELISAs can detect IMI in agricultural and environmental samples accurately and reliably. IMI residue has always been the focus of attention due to IMIs wide use. So, we believe that the specific peptide sequences and methods reported here are helpful to detect IMI residue. Further, unlike chemical hapten, peptide ligands can be prepared using a biological expression, so it is attractive to explore the peptide ligands to derive novel patterns of immunoassay, such as label-free immunoassay.In this study, thirty sequences of peptidomimetics and two sequences of anti-immunocomplex peptides for IMI were isolated from phage display peptide libraries, among which the anti-immunocomplex peptides were the first reported noncompetitive reagent for IMI. The peptidomimetic 1-9-H and anti-immunocomplex peptide 2-1-H that showed the best sensitivities were utilized to develop competitive and noncompetitive P-ELISAs. The IC


Anastomotic leakage is a life-threatening complication. Improvement of the anastomosis technique is needed, especially in patients with an inflamed edematous intestine. The aim of our study was to evaluate the safety and efficacy of an asymmetric figure-of-eight single-layer suture technique for intestinal anastomosis in pediatric patients.A total of 23 patients underwent intestinal anastomosis at the Department of Pediatric Surgery of Binzhou Medical University Hospital. Demographic characteristics, laboratory parameters, anastomosis time, duration of nasogastric tube placement, day of first postoperative bowel movement, complications, and length of hospital stay were statistically analyzed. The follow-up was conducted for 3–6 months after discharge.P = 0.036). The mean intestine anastomosis time in group 1 (18.83 ± 0.83 min) was less than that in group 2 . Patients in group 1 had an earlier first postoperative bowel movement . The duration of nasogastric tube placement in group 1 was shorter than that in group 2 . There was no significant difference in laboratory variables, complication occurrence, and length of hospital stay between the two groups.Patients were divided into two groups: the single-layer asymmetric figure-of-eight suture technique (group 1) and the traditional suture technique (group 2). Body mass index in group 1 was lower than in group 2 (14.43 ± 3.23 vs. 19.38 ± 6.74; The asymmetric figure-of-eight single-layer suture technique for intestinal anastomosis was feasible and effective. More studies are needed to compare the novel technique with the traditional single-layer suture. The exclusion criteria included the following: (1) patients who underwent anastomosis in the stomach or distal part of the rectum; and (2) with any of the above contraindications.Variables included age, sex, body mass index (BMI), laboratory data , length of postoperative hospital stay, time of anastomosis, duration of nasogastric tube placement, time of first postoperative bowel movement, and postoperative complications, including anastomotic bleeding, leakage, stricture, intra-abdominal abscess, pelvic collection, and abdominal distension. The time taken for the anastomosis (in minutes) was recorded from the beginning of the first stitch placement to the end of cutting extra suture material from the last stitch.An anastomotic leak was defined as follows: (1) body temperature >38°C, persistent abdominal pain, and tachycardia; (2) signs of peritonitis or intra-abdominal abscess confirmed by abdominal ultrasound; (3) non-absorbable material draining from a wound after oral administration; (4) elevated leucocyte count; and (5) visible disruption of the suture line during re-exploration , 19.Patients were divided into two groups: the single-layer asymmetric figure-of-eight suture technique group (group 1) and the traditional suture technique, i.e., single-layer interrupted sutures, group (group 2) , 20, 21.All patients were subjected to the same postoperative program, including antibiotics, administration of fluid, and placement of a nasogastric tube.via telephone or outpatient department interview for 3–6 months.The follow-up was conducted All procedures performed in the present study were in accordance with the ethical standards of the 1964 Helsinki Declaration. Written informed consent was obtained from the parents and/or legal guardians of the patients.t-test. Fisher's exact test and the Pearson chi-square test were used to analyze categorical data. A P-value <0.05 was considered statistically significant.The differences between the groups were analyzed using the SPSS Statistics version 26.0 software . Continuous data were analyzed using the Student's P = 0.036). The mean intestine anastomosis time in group 1 (18.83 ± 0.83 min) was significantly lower than in group 2 . The patients in group 1 had an earlier first postoperative bowel movement than those in group 2 . The duration of nasogastric tube placement in group 1 was significantly shorter than that in group 2 . No significant differences were found between the two groups with regard to sex distribution (P = 0.405), median age (P = 0.065), serum albumin level (P = 0.084), serum hemoglobin level (P = 0.054), C-reactive protein (P = 0.636), and white blood cell count (P = 0.230). Demographic and clinical characteristics are listed in A total of 23 pediatric patients who underwent anastomosis were assessed for eligibility, including 13 (56.52%) who underwent anastomosis with a single-layer asymmetric figure-of-eight suture technique  and 10 . DemograIleo-ileal, jejuno-jejunal, and portojejunal anastomoses were performed in groups 1 and 2. Major causes included gut malrotation, Meckel's diverticulum, perforation, gut or biliary atresia, duplication, small bowel obstruction, or neonatal necrotizing enterocolitis.No major postoperative complications were found in any of the children.Intestinal anastomosis is an essential surgical technique to restore intestinal continuity . In the Although the use of the traditional double-layer intestinal anastomosis or single-layer suture technique for intestinal anastomosis is controversial , the sinSeveral studies have reported that the early removal of the nasogastric tube and initiation of oral feeding can promote the recovery of intestinal function, shorten the length of the hospital stay, and prevent intestinal obstruction –28. In tOur results showed that the mean anastomosis time was significantly shortened in the asymmetric figure-of-eight single-layer suture technique group than in the single-layer interrupted sutures group, partly owing to the requirement of fewer knots . Time-saThis was a retrospective study. The sample size was relatively small, and the study was prone to the incomplete collection of individual data. Owing to the absence of a guideline to adhere to, the availability of intestinal anastomosis options may have been affected by the training or previous experience of pediatric surgeons. Furthermore, all patients were treated in a single medical center, and the results are not representative of other medical centers. Therefore, a multi-institutional trial with a large sample size determined using power analysis is required in the future.The asymmetric figure-of-eight single-layer suture technique was feasible and efficacious. It was superior to the traditional single-layer technique for intestinal anastomosis. Further studies are needed to confirm the preliminary findings.


In this work, polyethyleneimine (PEI)-grafted chitosan (Chi-g-PEI) was prepared for the fabrication of layer-by-layer (LBL) films for use in sustained-drug-delivery applications. Chi-g-PEI and polyacrylic acid (PAA) multilayer films were formed using the LBL technique. Methylene blue (MB) was used as a model drug for the investigation of loading and release capabilities of the LBL films. Characterizations of the synthesized copolymer were performed using Fourier-transform infrared spectroscopy (FTIR), Nuclear magnetic resonance spectroscopy (NMR), Thermogravimetric analysis (TGA), and X-ray Powder Diffraction (XRD) techniques, and the thickness of the LBL films was measured using Atomic force microscopy (AFM). The drug-loading and -release behaviors of the LBL films were assessed using a UV–visible spectrophotometer. The results showed that the loading capacity and release rate of MB were affected by ionic strength and pH. In addition, it was demonstrated that PEI-grafted chitosan is a good candidate for the assembling of LBL films for drug-delivery applications. The delivery of drugs is a vital part of medicine and healthcare services . Drug-deThe formation of multilayer thin films through the sequential adsorption of positively and negatively charged polymers on solid surfaces through electrostatic attraction in a layer-by-layer manner was first proposed by Iler in 1966 . Since tIt has limited applications due to low solubility in neutral and basic media. Grafting chitosan can improve its solubility and widen its applications, and because of that, it is the most attractive way to modify it . One of In this work, we designed PEI-grafted chitosan (Chi-g-PEI) using an imine reaction between low-molecular-weight PEI and periodate-oxidized chitosan to be able to use it for LBL multilayer film fabrication and for enhancing the loading capacity of chitosan. Modified chitosan was used to prepare LBL films with polyacrylic acid, and methylene blue was chosen as the model drug molecule to visualize the drug-loading and -release behaviors of the films. LBL Chi-g-PEI films can be used in the form of films on flat surfaces, nanoparticles, and nanocapsules for drug-delivery applications, such as nasal, ocular, or gastrointestinal applications ,20. TherChitosan , polyethylene imine (MW 25 kDa), potassium periodate, and sodium chloride were obtained from Sigma Aldrich . Methylene blue was obtained from Carlo Erba Reagent . Polyacrylic acid (PAA) (MW 150–170 kDa) was synthesized using radical polymerization as described in our previous study . 4 (0.01 M) in sodium acetate buffer (pH 4.5). The solutions were mixed after each solution was adjusted to 4 °C; the reaction was run for 48 h at room temperature and stopped by adding ethylene glycol (10% v/v). The final oxidized Cts was purified following a two-step dialysis procedure (Spectra/Por® membrane: MWCO = 3500) against NaCl  and with deionized water (pH 4.5). Secondly, PEI (20 mmol) was allowed to react with periodate-oxidized Cts solution (10 mmol) by stirring for 2 days at 4 °C. Then, the solution was treated with sodium borohydride (2 g NaBH4/g chitosan) and dialyzed against NaCl  (MWCO = 12–14 K) and against deionized water (pH 4.5) at 4 °C [PEI-grafted chitosan (Chi-g-PEI) was prepared according to the literature in two steps  22]. Fi. Fi22].  at 4 °C . After tv/v mixture of 98% H2SO4 and 30% H2O2) for 60 min and then rinsed with deionized water and dried. The multilayer films of Chi-g-PEI and PAA were prepared according to the following procedure The methylene blue-loaded LBL-film-coated glass slides were dipped in NaCl solutions at different NaCl concentrations and aqueous solutions with different pH values for release studies. A volume of 1000 μL of the sample was withdrawn from the outer phase; then, 1000 μL of solution was added to the outer phase during mechanical stirring. This process was repeated until the concentration value was constant.−1) were recorded with a Bruker spectrometer. A total of 64 spectra in the range of 4000–400 cm−1 were recorded with a resolution of 1 cm−1 and then averaged. 1H NMR spectra were recorded at room temperature in CDCl3  with a Bruker 400 MHz spectrometer at 300 or 400 MHz. X-ray powder diffraction (XRD) analyses were performed on a Rigaku SmartLab diffractometer  operated at 40 kV and 35 mA using Cu Kα radiation. The thermal stability was determined with a thermogravimetric analysis . The TGA thermograms were recorded using 5 mg of powder sample at a heating rate of 10 C/min in the temperature range of 30–800 °C in nitrogen atmosphere. Atomic force microscopy (AFM) was performed using Asylum Research MFP-3D-AS operated in tapping mode. To measure the thickness, the multilayered films were scratched using a scalpel, and the heights of the grooves were determined using AFM. A UV–visible analysis was carried out using a Shimadzu UV-2600 UV–Vis spectrometer.FTIR-ATR measurements  showed an absorption band at 1730 cm−1, which was attributed to the formed –C = O aldehyde groups. The third-spectrum band belonged to PEI-grafted chitosan. The main functional groups in the ethylene imine units of PEI are –CH2, −NH, and –NH2. The spectrum of PEI showed a peak at 1463 cm−1, which was attributed to the –CH2 group. The band at 1574 cm−1 corresponded to the vibration of the –NH group and the stretching vibration of the C-N groups of PEI [−1 were due to the asymmetric and symmetric stretching of the –CH2 groups [−1 corresponded to the –NH2 groups.l groups . The strs of PEI . The ban1H NMR spectroscopy (2CH2-).The modification of chitosan with PEI was checked using troscopy B. After θ = 10 and 2θ = 20, which were assigned to crystal forms I and II, respectively [The X-ray diffraction patterns of Chi and Chi-g-PEI are shown in ectively . After tectively .The thickness of LBL films was determined using AFM techniques. Multilayer LBL films of Chi-g-PEI and PAA were prepared on glass slides forming up to 10 layers, and the AFM images of the layers were measured. 10 multilayer films assembled on slides under various conditions. 10 films. 10 multilayer films assembled on slides in MB solutions with different ionic strengths. The loading of MB into LBL films reached adsorption equilibrium after 20 min for all different ionic strengths. The results showed that increasing the salt concentration affected the loading of MB solution. Therefore, the increase in NaCl concentration decreased the loading of MB. This could be related to the salting-out effect of the high salt concentration. Due to the high salt concentration, ionic bonds between two polymeric layers were disrupted. Thus, the layered structure was loosened, and layer separation occurred.During the loading process, the maximum absorption wavelength of MB, 668 nm, was used in subsequent experiments to calculate the amount of loaded MB on the (Chi-g-PEI/PAA)10-multilayer-film-coated slides. PAA is a weak polyelectrolyte, and the functional groups of PAA chains are carboxyl (–COOH) ones. The increase in pH dissociated the functional groups on the polymer chain. With pH below 4.5, the functional groups on the PAA chains were predominantly undissociated, because the pKa value of PAA is 4.5 [A is 4.5 . The pH A is 4.5 ,34. This10 films were evaluated at different NaCl concentrations, as shown in + in the solution penetrated into the films, and carboxylate groups (COO−) were protonated on the PAA chains. Thus, the MB molecule no longer interacted with the carboxylic acid groups (COOH) on the PAA chains at low pH values due to its positively charged structure [The effects of pH on the release rate of MB-loaded (Chi-g-PEI/PAA)tructure . This ca10 multilayer films were investigated. MB-loaded (Chi-g-PEI/PAA)10 films were immersed in solutions at different NaCl concentrations  and pH values . As shown in 10 multilayer films. As of a result of the electrostatic screening process, an increase in ionic strength led to the loosening of the film structure, thereby resulting in enhanced permeability and quick MB-release process from the films. Consequently, these results indicated that the MB release rate from films could be adjusted by modifying the ionic solution strength.The effects of the salt concentration on the MB release rate from (Chi-g-PEI/PAA)10 films was investigated. LBL-multilayer-film-assembled slides were dipped into 0.3 mg/mL MB solutions (0 mM NaCl) at pH 7.0 for 15 min. Then, the LBL-MB-loaded films were dipped in 150 mM NaCl solution at pH 7.0. This procedure was repeated for the desired number of times. As shown in 10 films. According to the results, no reasonable changes were observed after five times, and the film layers could reversibly load and release drug molecules.The reversibility of MB loading into and release from (Chi-g-PEI/PAA)10 multilayer films exhibited great reversibility in terms of the loading and release cycle of MB. Consequently, the release rate could be adjusted by adjusting pH and ionic strength. These results showed that (Chi-g-PEI/PAA)10 LBL multilayer films can be useful for the development of drug-delivery systems and other release applications.In this work, PEI-grafted chitosan was prepared and used for the formation of LBL films for drug-delivery applications. MB was used as a model drug to study drug delivery using Chi-g-PEI/PAA films. Both the ionic strength and pH value of the MB solution significantly affected MB loading into LBL films. The loading rate was decreased with the increase in ionic strength, further increasing as the pH was increased. Concerning the release process, the rate of release could be controlled via both the ionic strength and the pH value of the immersion solution. The acceleration of the release of MB from LBL films could be tailored via the administration of both high ionic strength and low pH. In addition, (Chi-g-PEI/PAA)


The etiology of radiation-induced pulmonary fibrosis is not clearly understood yet, and effective interventions are still lacking. This study aimed to identify genes responsive to irradiation and compare the genome expression between the normal lung tissues and irradiated ones, using a radiation-induced pulmonary fibrosis mouse model. We also aimed to map the mRNA alterations as a predictive model and a potential mode of intervention for radiation-induced pulmonary fibrosis. Thirty C57BL/6 mice were exposed to a single dose of 16 Gy or 20 Gy thoracic irradiation, to establish a mouse model of radiation-induced pulmonary fibrosis. Lung tissues were harvested at 3 and 6 months after irradiation, for histological identification. Global gene expression in lung tissues was assessed by RNA sequencing. Differentially expressed genes were identified and subjected to functional and pathway enrichment analysis. Immune cell infiltration was evaluated using the CIBERSORT software. Three months after irradiation, 317 mRNAs were upregulated and 254 mRNAs were downregulated significantly in the low-dose irradiation (16 Gy) group. In total, 203 mRNAs were upregulated and 149 were downregulated significantly in the high-dose irradiation (20 Gy) group. Six months after radiation, 651 mRNAs were upregulated and 131 were downregulated significantly in the low-dose irradiation group. A total of 106 mRNAs were upregulated and 4 downregulated significantly in the high-dose irradiation group. Several functions and pathways, including angiogenesis, epithelial cell proliferation, extracellular matrix, complement and coagulation cascades, cellular senescence, myeloid leukocyte activation, regulation of lymphocyte activation, mononuclear cell proliferation, immunoglobulin binding, and the TNF, NOD-like receptor, and HIF-1 signaling pathways were significantly enriched in the irradiation groups, based on the differentially expressed genes. Irradiation-responsive genes were identified. The differentially expressed genes were mainly associated with cellular metabolism, epithelial cell proliferation, cell injury, and immune cell activation and regulation. Radiotherapy is one of the primary treatments for cancer. More than two-thirds of all patients with cancer receive radiotherapy throughout the treatment procedure. Radiation-induced lung injury, which includes radiation-induced pneumonitis and pulmonary fibrosis, is a common and serious dose-limiting toxicity of thoracic radiotherapy and can be life-threatening. Radiation-induced pulmonary fibrosis (RIPF) is a late radiation-induced lung injury, and is generally incident 3 months post-irradiation, with symptoms ranging from mild dyspnea to chronic pulmonary insufficiency . Currentvia the TGFβ-R1/Smad3 signaling pathway  and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses to identify the function of the differentially expressed genes (DEGs) in the RIPF process.in vivo study protocols were approved by the Institutional Animal Care and Use Committee (NCC 2020A288) of the National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences .Thirty-six-week-old male C57BL/6 mice were acquired from HFK Bioscience Co., Ltd. . The mice were raised at the Experimental Animal Center of the Chinese Academy of Medical Sciences. The The mice were randomly divided into 3 groups (10 mice per group): control group (no irradiation), low-dose irradiation group, and high-dose irradiation group. The 10 mice in the low-dose group received 16 Gy single-dose thoracic radiation, while the mice in the high-dose group received thoracic radiation of 20 Gy. Radiation was administered using a Varian Unique-SN2242 unit at 300 cGy/min (1 m source to skin distance). The mice were anesthetized using Avertin (300 mg/kg), and were placed, in a supine position, on a fixing device and only the chest was exposed to radiation. Five mice from each group were euthanized 3 months after radiation. The process was repeated 6 months after radiation. After being euthanized, the left lobes of the lung tissues were extracted from the mice, frozen in liquid nitrogen, and stored at −80°C for RNA sequencing, while the right lobes were fixed in 4% formalin for pathological examination. For ethical considerations, mice with a weight loss of ≥20% were euthanized.To identify histological alterations caused by pulmonary fibrosis, histological analysis was performed as previously described . The lunTo compare the gene expression in lung tissues of normal mice and those of irradiated mice, apart from assessing the effects of different radiation doses on gene expression, tissue RNA was extracted and subjected to high-throughput sequencing using Illumina HiSeq. Total RNA was extracted from lung tissue samples using the TRIzol reagent (Invitrogen), according to the manufacturer’s instructions, as previously described . After cClean data were obtained by removing reads containing adapters, N bases, and low-quality reads from the raw data. The Q20, Q30, and GC values were calculated. All downstream analyses were based on high-quality clean data. The index of the reference genome was built using HISAT2 V2.0.5, and it was also used to align clean paired-end reads to the reference genome. We selected HISAT2 as the mapping tool because it can generate a database of splice junctions based on the gene model annotation file, producing better mapping results than other non-splice mapping tools. Feature Counts V1.5.0 was used to count the read numbers mapped to each gene. The expected number of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM) in each gene was calculated based on the length of the gene and the read count mapped to that gene.p-values were adjusted using Benjamini and Hochberg’s approach. Genes identified by DESeq2 to have an adjusted p-value ˂ 0.05 were denoted as differentially expressed. Enrichment analyses of these differentially expressed genes were performed. We used the clusterProfiler R package for GO function enrichment analysis, KEGG pathway enrichment analysis, and Reactome enrichment analysis of the differential genes.Analysis of differentially expressed genes in the low-dose and high-dose irradiation groups was performed using the DESeq2 R package (1.20.0), which provides statistical routines for determining differential expression using a model based on the negative binomial distribution. http://cibersortx.stanford.edu/) to analyze immune cell infiltration. The unpaired t-test was used to compare the infiltration levels of the immune cells in the two groups.CIBERSORT is a method for characterizing different types and relative abundances of immune cells in a mixed cell population. To estimate and compare the different proportions of infiltrated immune cells between the low-dose and high-dose irradiation groups, a gene expression matrix was uploaded to the CIBERSORT web portal , compared with the control group. In the high-dose irradiation group, 203 mRNAs were upregulated and 149 were downregulated significantly. The most upregulated mRNA in the low-dose irradiation group, after 3 months, was Gm10800, with a 153-fold change, while the most downregulated mRNA was Igkv4-74, with a 0.002-fold change. The most upregulated mRNA in the high-dose irradiation group was Ighv1-54, with a 103-fold change, and the most downregulated one was Nppa, with a 0.008-fold change. Six months after radiation therapy, in the low-dose irradiation group, 651 mRNAs were upregulated and 131 were downregulated significantly . In the high-dose irradiation group, 106 mRNAs were upregulated and 4 downregulated significantly. The most upregulated mRNA in the low-dose irradiation group after 6 months was Awat1, with a 121-fold change, and the most downregulated mRNA was Gm8941, with a 0.009-fold change. Igkv4-69 was the most upregulated mRNA in the high-dose irradiation group, with a 68-fold change, while Gm16499 was the most downregulated, with a 0.4-fold change. Volcano plots of the mRNAs in the 3 groups at the 2 time points depict their apparent variations . CorrespGO, KEGG, and Reactome enrichment analyses were performed to further investigate the potential functions of these DEGs, and the pathways involved with them.Staphylococcus aureus infection, complement and coagulation cascades, and the HIF-1 signaling pathway were enriched in the high-dose irradiation group , nuclear factor-κB (NF-κB), and TGF-β/Smad (Pathway enrichment analysis revealed that DEGs at 3 months were involved in the NOD-like receptor, TNF, and HIF-1 signaling pathways, all of which are injury- and inflammation-related pathways. NOD-like receptor protein 1 is called the first inflammasome and exerts its biological activity as an inflammasome complex . InflammF-β/Smad . In addiF-β/Smad . It has F-β/Smad . Based oAWAT1, MRGPRB2, IGKV4-69, IGHV3-1, and IGKV12-38. To gain insight into the distribution of immune cells, infiltration analysis was performed. It was observed that macrophages dominated at all times. The number of B cells increased with time and seemed to be related to the irradiation dose. Macrophages regulate tissue regeneration after injury. They may worsen tissue injury by producing reactive oxygen species and toxic mediators that disrupt cell metabolism, induce apoptosis, and exacerbate ischemic injury (We observed that at 6 months, immune-associated functions were significantly enriched, including myeloid leukocyte activation in the low-dose irradiation group and lymphocyte activation, lymphocyte proliferation, mononuclear cell proliferation, and immunoglobulin binding in the high-dose irradiation group. Some of the significantly dysregulated genes included c injury . On the c injury . Howeverc injury . B cell c injury . EvidencCD3 and TCR zeta chains, cytokine signaling in the immune system, and cellular senescence in the radiation groups were also enriched in the pathway analyses at 6 months. The former two are consistent with the functional analyses. An increasing body of evidence suggests that induction of senescence by radiation may play an important role in RIPF . It has Using RNA sequencing, we identified a large number of mRNAs at different stages of RIPF. These mRNAs were involved in cellular metabolism, cell injury, extracellular matrix function, epithelial cell proliferation, and immune system activation and regulation. Our results provide better understanding of the mechanism involved in the development of RIPF, which may be potential targets for preventing and treating RIPF.